Two preprints on protein evolution using cell-free translation systems have been posted on bioRxiv.
- Naohiro Terasaka
- 21 minutes ago
- 1 min read
The following two preprints have been opened on bioRxiv. They are collaborative research projects with Dr. Taro Furubayashi, a researcher at the Graduate School of Engineering, University of Tokyo.
Boosted cell-free gene expression for robust signal readout from a single-copy DNA template in microdroplets
Efficient cell-free evolution of RNA polymerases by droplet microfluidics
Traditional molecular evolution relies on DNA cloning, cell transformation, and culture processes, which are limited in speed and throughput.
In this study, we constructed a boosted PURE system capable of protein expression from several hundred fM of DNA and achieved single DNA detection in picoliter droplets.
Combining this Boosted PURE system with droplet screening enables completely cell-free protein evolution (PURE-FADS platform), which allows activity screening of approximately one million proteins in a single day.
Using this platform, we performed evolutionary engineering of SP6 RNA polymerase to improve its activity. Wild-type SP6 RNAP is highly salt-dependent, limiting its application in cell-free systems and intracellular systems. The SP6 RNAP-v3 evolved in this study exhibited high activity even in PURE systems under high salt conditions and functioned in mammalian cells.
Furthermore, we used the PURE-FADS platform to evolve a split SP6 RNAP and construct a proximity-dependent transcriptional biosensor (spSP6 RNAP-v4). Unlike the conventional split T7 system, it achieves near-zero background in vitro and maintains orthogonality with T7 RNAP. spSP6 RNAP-v4 can be used for protein interactions, antibody binding, RNA, and small molecule detection.
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